CN114660293A - SERS immunoassay kit for detecting mycotoxin exposure marker in blood plasma or urine - Google Patents
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Abstract
The invention provides a SERS (surface enhanced Raman scattering) immunoassay kit for detecting mycotoxin exposure markers in blood plasma or urine, which comprises a detection test strip and a detection reagent, wherein the detection test strip is provided with 3 detection lines, each detection line is coated with 2 different antigens, the detection reagent is six specific SERS nano-probes formed by combining monoclonal antibodies of six mycotoxin markers, reaction with two SERS nano-probes with different Raman markers on one detection line is realized by a SERS lateral flow immunosensing analysis method based on a competitive immunization principle, and the content of the mycotoxin markers in a sample is analyzed by SERS signal intensity generated by the nano-probes captured on the detection lines. The kit disclosed by the invention has the advantages that the detection limit of each mycotoxin marker in the blood plasma and urine extraction solution is between 0.0022 ng/mL and 0.21ng/mL, the accuracy and the precision are high, and the kit can be used for quickly detecting the mycotoxin markers in the blood plasma and urine in a farm or a basic laboratory.
Description
Technical Field
The invention relates to the technical field of Raman spectrum detection and competitive immunoassay, in particular to a kit for detecting a mycotoxin exposure marker in blood plasma or urine.
Background
Mycotoxins are common hazard factors in livestock and poultry production, particularly live pig breeding, and mainly comprise aflatoxin, zearalenone, vomitoxin, fumonisin, ochratoxin, T-2toxin and the like. The mycotoxin can produce obvious poisoning symptoms such as anorexia, vomit, jaundice, diarrhea, reproductive disorders and even acute death on livestock and poultry, so that the mycotoxin pollution is prevented and controlled in the breeding production, and the method has important significance for guaranteeing the healthy breeding of the livestock and poultry.
At present, mycotoxin is mainly prevented and controlled in the breeding process by monitoring the content of mycotoxin in feed and adding a detoxifying agent according to actual conditions to relieve the toxic effect of mycotoxin. Due to serious and uneven distribution of mycotoxins in feed and feed raw materials, sampling and analysis errors are easily generated, and due to the existence of concealed mycotoxins, the actual content of mycotoxins in a sample is often underestimated by the result of conventional analysis. Secondly, the feed and feed materials may grow mold contamination during processing, transportation, storage and feeding in the farm to produce mycotoxins. Therefore, analysis of the mycotoxin content in the feed does not accurately reflect the actual exposure level of mycotoxins in the livestock and poultry. Researches show that the concentration of main mycotoxin metabolic markers in livestock and poultry is highly related to ingested mycotoxin, and main concealed mycotoxins can be converted into free forms in vivo, so that the mycotoxin marker content in biological samples such as plasma, urine, feces and the like can be analyzed, and the mycotoxin exposure condition of pig bodies can be reflected more directly and accurately.
The effectiveness of the mycotoxin detoxification agent is generally evaluated by an in vitro test, the effect of the mycotoxin detoxification agent in vivo cannot be truly reflected, and an evaluation means combining in vivo and in vitro needs to be established urgently to standardize the application of the mycotoxin detoxification agent product. An analysis method for the mycotoxin exposure marker in biological samples such as plasma and urine is researched and established, and a technical means can be provided for mycotoxin exposure evaluation and mycotoxin detoxication agent effectiveness evaluation in livestock and poultry bodies.
The mycotoxin markers in livestock and poultry have various types and low content, the detection requirements of the mycotoxin markers are difficult to meet by a conventional analysis method, and the mycotoxin markers are mainly determined by a liquid chromatography mass spectrometry at present. The method has high sensitivity, accuracy and precision, but needs expensive instruments and equipment, professional technicians and complex sample pretreatment, so the method is not suitable for rapid detection of mycotoxin markers in primary laboratories and field environments. The method has the advantages of high detection speed, low detection cost and the like, but has low detection sensitivity and lacks multi-component analysis capability, and cannot meet the detection requirement of low-level mycotoxin combined exposure. Therefore, research and development of a rapid analysis method with higher sensitivity and stronger multi-component detection capability are urgently needed, and a more economic and efficient technical means is provided for mycotoxin pollution monitoring of a culture site.
Immunosensing analysis based on Surface Enhanced Raman Scattering (SERS) is a novel rapid detection technology combining SERS markers and antigen-antibody immunoreaction, has the advantages of high detection speed, high sensitivity, strong multicomponent detection capability and the like, and can meet the detection requirement of mycotoxin metabolic markers in biological samples. SERS tags have several advantages: firstly, the Raman spectrum has high molecular characteristics and narrow spectral peak which is only 1/10-1/100 of the fluorescence spectrum, so that the spectral peak overlap among different molecules can be reduced, and the Raman spectrum is suitable for multi-element labeling immunoassay; secondly, in SERS multi-component detection, different Raman active molecules can be excited by single-wavelength exciting light, so that the requirements on light source configuration are low; thirdly, SERS signals are not easy to generate self-quenching phenomenon, and signal intensity can be improved by increasing the number of Raman active molecules, so that the sensitivity of immunoassay is improved. SERS-based lateral flow immunosensing analysis has attracted extensive attention and research in recent years, but most of the current research is single-component analysis, the advantages of SERS multi-label analysis and lateral flow immunosensing multi-detection-line mode cannot be fully exerted, and the detection requirement of synchronous analysis of various common mycotoxin markers cannot be completely met.
Disclosure of Invention
The invention aims to provide an SERS immunoassay kit for detecting mycotoxin exposure markers in blood plasma or urine, which is used for rapidly, accurately and sensitively detecting aflatoxin M in urine or blood plasma on site1(AFM1) Ochratoxins (OTAs), Zearalenone (ZEA), T-2toxin (T-2), fumonisins (FB1) and vomitoxin (DON).
The immunosensing analysis method adopted by the patent is a lateral flow immunosensing analysis method based on Surface Enhanced Raman Scattering (SERS). According to the method, 5' -dithiobis (2-nitrobenzoic acid) (DTNB) and p-mercaptobenzoic acid (MBA) are respectively used as Raman markers to mark Au @ Ag nano particles, and then the Au @ Ag nano particles are respectively combined with the monoclonal antibodies of the six mycotoxin markers to form six specific SERS nano probes. The carrier of the method is a lateral flow immunochromatographic test strip which comprises a sample pad, a nitrocellulose membrane and an absorption pad. 3 detection lines are arranged on the nitrocellulose membrane, each detection line is mixed with 2 different antigens, and the detection lines can react with two different SERS nanoprobes simultaneously, so that six different mycotoxin exposure markers can be detected simultaneously on one test strip.
The invention firstly provides a kit for detecting mycotoxin exposure markers in blood plasma or urine, which comprises a detection test strip and a detection reagent; the immune test strip is a lateral flow immunochromatography test strip, and comprises 3 detection lines, and each detection line is coated with 2 different antigens in a mixed manner; the antigens are aflatoxin M1, ochratoxin, zearalenone, T-2toxin, fumonisin B1 and vomitoxin respectively; the detection reagent is an SERS nano probe which is formed by coupling an Au @ Ag core-shell nanoparticle compound labeled with different Raman molecular markers with an aflatoxin M1 monoclonal antibody, an ochratoxin monoclonal antibody, a zearalenone monoclonal antibody, a T-2toxin monoclonal antibody, a fumonisin B1 monoclonal antibody and an vomitoxin monoclonal antibody.
The detection test strip is provided with a quality control line, and the quality control line is sprayed with a secondary antibody-nano gold compound.
Preferably, in 3 detection lines on the detection test strip, each detection line is coated with 2 different antigens in a mixed manner, and the combination mode is as follows:
aflatoxin M1An antigen and ochratoxin antigen;
zearalenone antigen and T-2toxin antigen;
fumonisins B1Antigens and vomitoxin antigens.
Preferably, aflatoxin and ochratoxin antigens are coated on a detection line far away from the sample pad, zearalenone and T-2toxin antigens are coated on the position of the middle detection line, and fumonisin and vomitoxin are coated on the position of the detection line close to the sample pad; namely, if the sample pad is arranged on the left side of the detection test strip, 3 detection lines from left to right are respectively a detection line coated by fumonisin and vomitoxin, a detection line coated by zearalenone and T-2toxin antigen, and a detection line coated by aflatoxin and ochratoxin antigen.
Further preferably, in the detection reagent,
with aflatoxin M1Raman markers of SERS nano probes coupled with the monoclonal antibody and the ochratoxin monoclonal antibody are different;
the Raman marker is different from the Raman marker of the SERS nano probe coupled with the zearalenone monoclonal antibody and the T-2toxin monoclonal antibody;
with fumonisins B1The Raman markers of the SERS nano-probe coupled with the monoclonal antibody and the vomitoxin monoclonal antibody are different.
The Raman marker is 5, 5-dithiobis-2-nitrobenzoic acid (DTNB), p-mercaptoaniline (PATP), p-mercaptobenzoic acid (MBA) or 2,2' -bipyridyl (Bipy).
More preferably, in the detection reagent, aflatoxin M is reacted with1Raman markers of SERS nanoprobes coupled with the monoclonal antibody and the Ochratoxin (OTA) monoclonal antibody are DTNB and MBA respectively;
raman markers of the SERS nanoprobes coupled with the zearalenone monoclonal antibody and the T-2toxin monoclonal antibody are DTNB and MBA respectively;
with fumonisins B1Raman markers of the SERS nanoprobes coupled with the monoclonal antibody and the vomitoxin monoclonal antibody are DTNB and MBA respectively.
Through experimental screening, the AFM1The sensitivity generated by the SERS nano-probe prepared by coupling the monoclonal antibody and the ZEA monoclonal antibody with DTNB-Au @ Ag NPs is higher than that generated by the SERS nano-probe prepared by coupling the MBA-Au @ Ag NPs compound. Comprehensively considering the original setting condition of the coating on the detection line, and finally determining the AFM1、ZEA、FB1The monoclonal antibody and DTNB-Au @ Ag NPs are coupled to prepare the SERS nano-probe, and the OTA, T-2 and DON monoclonal antibodies and MBA-Au @ Ag NPs are coupled to prepare the SERS nano-probe, so that the detection sensitivity of the kit is optimal.
In the kit provided by the invention, the particle size of the Au @ Ag core-shell nanoparticle compound is 48-56nm, and preferably 52 nm.
In the kit provided by the invention, in the detection reagent, the concentrations of SERS nano-probes of six mycotoxins are OD520nm1.0, mixing at the volume ratio of 1:2:1:3:1:2, the total volume is 200 mu L, and placing in a plate hole of an enzyme label plate for freeze-drying for later use.
The preparation method of the Au @ Ag core-shell nanoparticle compound comprises the following steps: 60 μ L of 200mM ascorbic acid and 15 μ L of 200mM AgNO were mixed at room temperature3Adding the mixture into a 10mL container of Au NPs with the particle size of about 30nm, and continuing to react for 30min under the stirring condition; 60 μ L of 200mM ascorbic acid and 15 μ L of 200mM AgNO were added3Reacting for 30 min; au @ Ag core-shell nanoparticle composites with the particle size of 48-56nm are prepared sequentially through 4 cycles, and the obtained particle composites are respectively centrifuged for 20min and redissolved in 10mL of ultrapure water.
The preparation steps of the SERS nanoprobe are as follows: mixing 10mLAu @ Ag core-shell nanoparticle complex with 500 μ L of 0.2M pH 8.5 boric acid buffer, adding 300 μ L of 1mM DTNB or 1mM MBA; the solutions are respectively slightly shaken at room temperature for reaction for 30min, centrifuged, redundant Raman molecules in supernatant are removed, and 10mL of 2.0mM boric acid buffer solution with pH of 8.5 is used for redissolving; respectively adding six mycotoxin antibodies into DTNB-Au @ Ag core-shell nanoparticle compound or MBA-Au @ Ag core-shell nanoparticle compound solution, and reacting for 1h under slow stirring; respectively adding 200 μ L of 1% PVP dissolved in 2.0mM boric acid buffer solution with pH of 8.5 for treatment, and masking bare sites on the surfaces of the nanoparticles to terminate the reaction; this suspension was centrifuged at 3,400rpm for 10min, the supernatant removed, and the centrifugation was repeated twice after reconstitution, and the pellet was resuspended in 0.01M, pH 7.4 phosphate buffer containing 1% BSA.
The SERS lateral flow immunosensing analysis principle based on the kit is shown in figure 1. Firstly, respectively spraying three groups of mixed antigens on different positions of a nitrocellulose membrane as detection lines, and spraying a goat-anti-mouse secondary antibody on the nitrocellulose membrane as a quality control line; SERS nano-probes capable of identifying six mycotoxin markers are arranged in the micropores. If the sample does not contain any mycotoxin marker, a specific SERS nano probe is combined with the coating antigen on the nitrocellulose membrane, visible colors appear on three detection lines, and a strong SERS signal is generated under the excitation of laser (the characteristic waveband of the nano probe marked by DTNB is 1332 cm)-1The characteristic wave band of the MBA-labeled nano probe is 1589cm-1). However, when the sample contains a certain mycotoxin marker, the free analyte in the sample will compete with the coating antigen on the detection line for the recognition site on the specific SERS nanoprobe, and the coating antigen on the corresponding detection line on the nitrocellulose membrane will react with less specific SERS nanoprobes, so the SERS signal of the specific band on the detection line will be correspondingly reduced. If the sample contains a certain mycotoxin marker with a high content, the reaction between the specific SERS nano-probe and the coating antigen can be completely blocked, so that SERS signals of corresponding wave bands cannot appear on the detection line. Meanwhile, the cellulose nitrate membrane is provided with a quality control line, when the operation is normal, the second antibody on the quality control line can interact with the SERS nano probe, and therefore, the quality control line can generate a strong SERS signal no matter whether the sample is negative or positive. During quantitative analysis, the SERS signal intensity of the characteristic wave band of the unknown sample at the detection line is measured and substituted into the corresponding standard curve, and then a certain kind of the sample can be calculatedLevels of mycotoxin markers.
The invention provides a SERS (surface enhanced Raman scattering) lateral flow immunosensing analysis method for detecting mycotoxin exposure markers in blood plasma or urine, wherein the mycotoxin is aflatoxin M1Ochratoxin, zearalenone, T-2toxin and fumonisin B1And vomitoxin; the method is characterized by comprising the following steps:
(1) pretreatment of a sample to be detected:
a) plasma sample pretreatment: taking 0.3mL of a plasma sample, adding 0.7mL of acetonitrile, fully whirling, mixing uniformly, centrifuging at 8000rpm for 10min, taking 0.5mL of supernatant, fully mixing uniformly with 2mL of PBS (phosphate buffer solution) solution containing 0.5% Triton-100 and 0.5% BSA (bovine serum albumin), and taking 200 mu L of the mixture for SERS lateral flow immunosensing analysis; or
b) Pretreatment of a urine sample: centrifuging the urine sample at 6,000rpm for 5 minutes, uniformly mixing 0.5mL of supernatant with 0.5mL of PBS (phosphate buffer solution) containing 1% Triton and 1% BSA, and taking 200 mu L of supernatant for SERS lateral flow immunosensing analysis;
(2) the kit is used for detecting a sample to be detected: adding 200 mu L of sample solution into a detection reagent, uniformly mixing, incubating at room temperature for 3min, immersing the immune test strip into the incubated reagent, reacting for 10min, taking out the test strip, drying, and collecting SERS signals at the detection line by using a Raman spectrometer; the spectrometer parameters in the experiment were set as follows: the excitation light source adopts a He-Ne laser, the excitation wavelength is 785nm, the laser intensity is 20%, and the signal acquisition time is 5 s; separately record the DTNB labeled nanoprobes for 1332cm-1And 1589cm of MBA-labeled nano probe-1Signal strength of the band.
The kit for detecting the mycotoxin exposure marker in the blood plasma or urine adopts a competitive immunoassay principle, and finally can analyze the content of the mycotoxin exposure marker in a sample through the SERS signal intensity generated by the SERS nano-probe captured on a detection line. The detection limit of each mycotoxin exposure marker in the blood plasma and urine extraction solution measured by the method is verified to be between 0.0022 ng/mL and 0.21ng/mL, which is far lower than that of most reported instrument analysis and immunoassay, and meanwhile, the accuracy and precision of the method meet the requirements of quantitative or semi-quantitative analysis. The method can be used for exposure evaluation of the mycotoxin in the on-site environment, has high accuracy and high sensitivity for detecting the content of the mycotoxin in animal plasma or urine, is time-saving and labor-saving, can be used for detecting the mycotoxin exposure markers in the plasma and urine in a farm or a basic laboratory, and has wide application value and application prospect.
Drawings
FIG. 1 is a schematic diagram of SERS lateral flow immunosensing analysis of mycotoxin markers.
FIG. 2 shows the effect of Au @ Ag nanoparticle size on the intensity of DTNB signature (left panel) and MBA signature (right panel).
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means and conventional detection methods well known to those skilled in the art. All reagent consumables in the examples are commercially available unless otherwise specified.
The following examples all adopt a method of repeating the experiment 5 times, and the results show that the data of the parallel experiment results of each batch of the repeated experiment are not significantly different, and the final average value is listed in the examples.
The six mycotoxin monoclonal antibodies are obtained in the laboratory through the steps of mouse immunization, cell fusion, hybridoma screening, ascites preparation, antibody purification and the like by a conventional method. Six mycotoxin Antigens (AFM) mentioned in the examples1、ZEA、FB1OTA, T-2, DON) antigens, prepared according to the methods disclosed in the prior art.
AFM1Antigen synthesis methods references: chu, F.S, Ueno, I.,1977.Production of antibiotics against aflatoxin B1.appl. environ. Microbiol.33, 1125-1128.
ZEA antigen synthesis method reference: thouvenot, D., Morfin, R.F.,1983.Radioimmunoassay for zearalenone and zearalanol in human serum: production, properties, and use of cosmetic agents, Appl. environ.Microbiol.45, 16-23.
FB1Antigen synthesis methods references: yu, F.Y., Chu, F.S.,1996.Production and characterization of antibiotics against fumonisin B1, J.food prot.59, 992-997.
DON antigen synthesis method references: maragos, C.M., Mccormick, S.P.,2000.Monoclonal Antibodies for the Mycotoxin Deoxynivanols and 3-Acetyl-Deoxynivanols food Agr. Immunol.12, 181-192.
T-2 antigen Synthesis reference: chu, F.S., Grossman, S., Wei, R., Mirocha, C.J.,1979.Production of Antibody Against T-2Toxin. appl.environ. Microbiol.37, 104-108.
OTA antigen synthesis method references: liu, b.h., Tsao, z.j., Wang, j.j., Yu, f.y.,2008.Development of a monoclonal antibody against ochratoxin a and its application in enzyme-linked immunological assay and gold nanoparticles immunological strip.
Example 1 preparation, identification and particle size selection of Au @ Ag nanoparticles
Preparation of Au @ Ag NPs is described by Blanco-Covi-n et al: first, 60. mu.L of Ascorbic Acid (AA) (200mM) and 15. mu.L of AgNO were mixed at room temperature3(200mM) was added to 10mL of AuNPs solution having a particle size of about 30nm, and the reaction was carried out for 30min with stirring. Then 60. mu.L of AA (200mM) and 15. mu.L of AgNO3(200mM) were added for 30min (second cycle). The thickness of the silver shell of the Au @ Ag NPs increases with the increase of the adding times. Au @ Ag NPs were prepared according to the different cycle numbers, with cycle numbers of 1, 2, 4, respectively, and the resulting particles were centrifuged for 20min, respectively, and the particles were redissolved in 10mL of ultrapure water again. Finally, the prepared nanoparticles with the particle diameters of 48-56nm are identified and can be found to contain obvious core-shell structures, which indicates that the Au @ Ag NPs are successfully synthesized.
The particle size of the Au @ Ag nanoparticles can have a significant influence on the SERS signal intensity, and as can be seen from FIG. 2, when the particle size of the nanoparticles is increased from 32nm to 40nm, the DTNB and MBA characteristic signal intensities are increased and continuously increased to 52nm, and the corresponding SERS signals are continuously increased. However, in the experimental process, it is found that when the particle size of the nanoparticle is larger than 52nm, the nanoparticle is easy to generate a condensation phenomenon in the process of preparing the SERS nanoprobe, or the prepared SERS nanoprobe is easy to generate a condensation phenomenon in the long-term storage process, the nanoparticle with the particle size of about 52nm can generate a strong enough Raman signal, and meanwhile, the stability can be kept in the processes of preparing the SERS nanoprobe and storing for a long time, so that the 52nm Au @ Ag nanoparticle is finally selected to prepare the SERS nanoprobe.
Example 2 preparation and optimization of SERS nanoprobes
The preparation method of the SERS nanoprobe comprises the following specific steps: first, 10 mM of prepared @ Ag NPs were mixed with 500. mu.L of boric acid buffer (0.2M, pH 8.5), and 300. mu.L of 1mM of 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB) or 1mM of p-mercaptobenzoic acid (MBA) was added. The solutions were reacted with each other at room temperature with gentle shaking for 30min, centrifuged, and the excess raman molecules in the supernatant were removed, followed by reconstitution with 10mL of a boric acid buffer (2.0mM, pH 8.5). Then, six mycotoxin antibodies were added to the solutions of DTNB-Au @ Ag NPs or MBA-Au @ Ag NPs, respectively, and reacted for 1 hour with slow stirring. Finally, 200. mu.L of 1% PVP in boric acid buffer (2.0mM, pH 8.5) was added to stop the reaction by masking the exposed sites on the surface of the nanoparticles. This suspension was centrifuged at 3,400rpm for 10min, the supernatant removed, and the centrifugation was repeated twice after reconstitution, and the pellet was resuspended in 1% BSA in phosphate buffer (0.01M, pH 7.4). And finally, mixing the SERS nano-probes of the six mycotoxins according to the optimal proportion, transferring the mixture to an enzyme-labeled plate hole, and freeze-drying the mixture for later use.
In the preparation process of the SERS nano-probe, six mycotoxin monoclonal antibodies are respectively coupled with DTNB-Au @ Ag NPs or MBA-Au @ Ag NPs compounds, then the prepared SERS nano-probe is adopted to carry out immunoassay, the sensitivity is measured, and the result (Table 1) shows that AFM (atomic force microscopy) is adopted1Sensitivity ratio generated by SERS nano-probe prepared by coupling monoclonal antibody and ZEA monoclonal antibody with DTNB-Au @ Ag NPs and MBA-Au @ Ag NPs compoundThe sensitivity of the SERS nano-probe prepared by coupling is high, and the sensitivity of other antibodies is equivalent to that of the SERS nano-probe prepared by two compounds. Comprehensively considering the original setting condition of the coating on the detection line, and finally determining the AFM1、ZEA、FB1And (3) coupling the monoclonal antibody with DTNB-Au @ Ag NPs to prepare an SERS nano-probe, and coupling the OTA, T-2 and DON monoclonal antibodies with MBA-Au @ Ag NPs to prepare the SERS nano-probe.
TABLE 1 comparison of detection limits of monoclonal antibodies with DTNB and MBA labeled nanoprobes
| DTNB labeled nanoprobe | MBA (moving bed antigen) labeled nano probe | |
| AFM1Monoclonal antibody | 0.0021ng/mL | 0.0032ng/mL |
| ZEA monoclonal antibody | 0.0091ng/mL | 0.013ng/mL |
| FB1Monoclonal antibody | 0.183ng/mL | 0.188ng/mL |
| OTA monoclonal antibody | 0.0171ng/mL | 0.0174ng/mL |
| T-2 monoclonal antibody | 0.0142ng/mL | 0.0144ng/mL |
| DON monoclonal antibody | 0.093ng/mL | 0.091ng/mL |
Example 3 optimization and location of mycotoxin antigen combinations
This example compares six mycotoxin Antigens (AFB)1-BSA, OTA-BSA, T2-OVA, ZEA-BSA, DON-BSA and FB1OVA) at the positions of 3 detection lines of nitrocellulose respectively, and the results (Table 2) show that, in addition to fumonisin antigen, the sensitivity obtained by immobilizing other mycotoxin antigens on the detection line (detection line 1) far away from the sample pad position is higher than the sensitivity obtained on the detection line (detection line 3) near the sample pad position. Meanwhile, considering that the aflatoxin and ochratoxin in blood plasma and urine are generally the lowest, the zearalenone and T-2toxin are slightly higher, and the fumonisin and vomitoxin are generally relatively higher in concentration, the invention fixes the aflatoxin and ochratoxin antigens on a detection line (detection line 1) far away from a sample pad, fixes the zearalenone and T-2toxin antigens on the position of a middle detection line (detection line 2), and fixes the fumonisin and vomitoxin on the position of a detection line (detection line 3) close to the sample pad.
TABLE 2 comparison of detection limits generated by different detection line positions coated with mycotoxin antigens
| |
Detection line 2 | Detection line 3 | |
| AFB1-BSA | 0.0022ng/mL | 0.0026ng/mL | 0.0031ng/mL |
| OTA-BSA | 0.0173ng/mL | 0.0181ng/mL | 0.0189ng/mL |
| T2-OVA | 0.0134ng/mL | 0.0143ng/mL | 0.0155ng/mL |
| ZEA-BSA | 0.0083ng/mL | 0.0090ng/mL | 0.0098ng/mL |
| DON-BSA | 0.078ng/mL | 0.085ng/mL | 0.094ng/mL |
| FB1-OVA | 0.181ng/mL | 0.184ng/mL | 0.183ng/mL |
EXAMPLE 4 preparation of an Immunity lateral flow test strip
(1) Preparation of nitrocellulose membranes
Subjecting aflatoxin M1(AFM1) And mixed antigen of Ochratoxin (OTA), mixed antigen of Zearalenone (ZEA) and T-2toxin (T-2), Fumonisin (FB)1) And vomitoxin (DON), a goat anti-mouse secondary antibody (0.5mg/mL) were dissolved in a carbonic acid buffer solution (0.05M, pH 9.5), and then 3 detection lines and 1 quality control line were sprayed on the nitrocellulose membrane at a speed of 1. mu.L/cm and at an interval of 3.0mm using a streaking apparatus, respectively. Finally, the nitrocellulose membrane was dried at 37 ℃ for 6h and dry sealed at room temperature for use.
(2) Assembly of test strips
Fixing the nitrocellulose membrane coated with the mixed envelope antigen and the goat anti-mouse second antibody at the center of the bottom plate, fixing the sample pad at one end, overlapping the nitrocellulose membrane at the center for 2-4 mm, and fixing the absorption pad at the other end, overlapping the nitrocellulose membrane for 2-4 mm. And finally, cutting the assembled bottom plate into test strips with the width of 4mm, and sealing for later use.
Example 5 establishment of SERS lateral flow immunosensing assay for detection of six mycotoxin exposure markers in urine or plasma
(1) Plasma sample pretreatment: taking 0.3mL of a plasma sample, adding 0.7mL of acetonitrile, fully whirling and mixing uniformly, centrifuging at 8000rpm for 10min, taking 0.5mL of supernatant, fully mixing with 2mL of PBS (phosphate buffer solution) containing 0.5% Triton-100 and 0.5% BSA, and taking 200 mu L of the mixture for SERS lateral flow immunosensing analysis.
(2) Pretreatment of a urine sample: urine samples were centrifuged at 6,000rpm for 5 minutes, 0.5mL of the supernatant was mixed with 0.5mL of 1% Triton and 1% BSA in PBS, and 200 μ L was used for SERS lateral flow immunosensing analysis.
(3) The determination process comprises the following steps: adding 200 μ L sample solution into microporous plate, mixing with lyophilized nanoprobe in the well, incubating at room temperature for 3min, soaking the test strip in the sample well, reacting for 10min, allowing the solution to move toward the absorption pad, and detectingSpecific binding occurs at the line and the quality control line. And after the reaction is finished, taking out the test strip for drying, and collecting SERS signals at the detection line by using a Raman spectrometer. The spectrometer parameters in the experiment were set as follows: the excitation light source adopts a He-Ne laser, the excitation wavelength is 785nm, the laser intensity is 20%, and the signal acquisition time is 5 s. Record 1332cm each-1(DTNB-labeled nanoprobe) and 1589cm-1(MBA-labeled nanoprobe) band signal intensity.
(4) Preparation of the Standard Curve
And taking negative plasma and urine samples, and processing according to the sample pretreatment mode. And respectively adding a proper amount of mycotoxin standard solution into the negative sample extracting solution to prepare a series of mixed standard working solutions. Wherein AFM1Are 0, 0.0027, 0.0082, 0.025, 0.074, 0.22, 0.67 and 2.0ng/mL respectively, OTA is 0, 0.027, 0.082, 0.25, 0.74, 2.22, 6.67 and 20ng/mL respectively, ZEA is 0, 0.017, 0.049, 0.15, 0.44, 1.33, 4.0 and 12ng/mL respectively, T-2 is 0, 0.017, 0.049, 0.15, 0.44, 1.33, 4.0 and 12ng/mL respectively, FB1Are 0, 0.27, 0.82, 2.5, 7.4, 22.2, 66.7 and 200ng/mL, and the concentration of DON is 0, 0.14, 0.41, 1.23, 3.70, 11.1, 33.3 and 100ng/mL, respectively. 3 replicates of each mixed standard solution were tested according to the above test procedure. Calculating the ratio (B/B) of the intensity of the Raman signal generated at each concentration to the intensity of the Raman signal generated by the negative sample0) As an ordinate, a standard curve was plotted with a natural logarithm of the concentration of the standard solution (lnC) as an abscissa. The concentration at which the signal intensity was reduced by 10% compared to that of the negative sample was used as the detection limit. The formulas and detection limits of the standard curves for each mycotoxin were determined as shown in tables 3 and 4.
TABLE 3 Standard Curve formula and detection limits for mycotoxins in plasma samples
| Formula of standard curve | R2 | A detection limit; ng/mL | |
| AFM1 | y＝-0.114ln(x)+0.2033 | 0.9925 | 0.0022 |
| OTA | y＝-0.107ln(x)+0.4638 | 0.9912 | 0.0169 |
| ZEA | y＝-0.1091n(x)+0.3885 | 0.9916 | 0.0092 |
| T-2 | y＝-0.117ln(x)+0.4033 | 0.9947 | 0.014 |
| FB1 | y＝-0.114n(x)+0.7039 | 0.9938 | 0.18 |
| DON | y＝-0.132ln(x)+0.5821 | 0.9908 | 0.090 |
TABLE 4 Standard Curve formula and detection limits of mycotoxins in urine samples
| Formula of standard curve | R2 | A detection limit; ng/mL | |
| AFM1 | y＝-0.113ln(x)+0.2073 | 0.9925 | 0.0022 |
| OTA | y＝-0.108ln(x)+0.4762 | 0.9912 | 0.0198 |
| ZEA | y＝-0.11ln(x)+0.3969 | 0.9916 | 0.0103 |
| T-2 | y＝-0.118ln(x)+0.3974 | 0.9947 | 0.014 |
| FB1 | y＝-0.113ln(x)+0.7249 | 0.9938 | 0.21 |
| DON | y＝-0.131ln(x)+0.592 | 0.9908 | 0.095 |
(5) Determination of method accuracy and precision (addition recovery test)
Adding mycotoxin standard solutions with different concentrations into blank plasma and urine samples, performing standard addition recovery determination, and performing 4 parallels on each concentration to evaluate the accuracy and precision of the immunosensing analysis method. And during measurement, a Raman spectrometer is adopted to collect the Raman signal intensity of the detection line, and the Raman signal intensity is substituted into a corresponding standard curve to calculate the concentration of each mycotoxin marker in the sample and calculate the recovery rate and the coefficient of variation. The results are shown in table 5, the recovery rate of the mycotoxin marker is between 82.4% and 118.6% at each addition concentration, and the coefficient of variation is less than 20%, which indicates that the accuracy and precision of the SERS lateral flow immunosensor analysis substantially meet the requirements of quantitative and semi-quantitative analysis.
Table 5SERS lateral flow immunosensing assay mycotoxin exposure markers add recovery and coefficient of variation (n ═ 4)
EXAMPLE 6 actual sample testing
Collecting 3 parts of each pig plasma and urine sample from different pig farms, pretreating and measuring the pig plasma and urine samples together with negative samples by the method, obtaining the corresponding Raman signal intensity of each detection line, and dividing the Raman signal intensity by the corresponding signal intensity of the negative samples to obtain a signal relative ratio (B/B)0) And substituting the concentration into a standard curve, calculating to obtain the concentration in the extraction solution, and multiplying the concentration by the corresponding dilution times to obtain the concentration of the corresponding mycotoxin exposure metabolic marker in the sample. The measurement results of the actual samples are shown in Table 6.
TABLE 6 SERS immunosensory analysis results (ng/mL) of actual samples
| Sample type and number | AFM1 | OTA | ZEA | T-2 | | DON |
| Plasma | ||||||
| 1 | -- | -- | 2.96 | -- | -- | 3.18 |
| Plasma 2 | -- | 0.71 | 3.33 | -- | -- | 2.74 |
| Plasma 3 | 0.63 | 0.92 | 7.83 | 1.24 | 3.21 | 12.9 |
| |
-- | -- | 0.74 | -- | -- | 1.25 |
| Urine 2 | -- | 0.08 | 0.64 | -- | -- | 0.46 |
| Urine 3 | 0.11 | 0.17 | 1.11 | 0.13 | 0.52 | 5.2 |
While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain modifications and improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. The SERS immunoassay kit for detecting the mycotoxin exposure marker in blood plasma or urine is characterized by comprising a detection test strip and a detection reagent; the immune test strip is a lateral flow immunochromatography test strip, and comprises 3 detection lines, and each detection line is coated with 2 different antigens in a mixed manner; the antigens are respectively aflatoxin M1Ochratoxin, zearalenone, T-2toxin and fumonisin B1And vomitoxin; the detection reagent is an SERS nano probe which is an Au @ Ag core-shell nanoparticle compound for marking different Raman molecular markers and aflatoxin M respectively1Monoclonal antibody, ochratoxin monoclonal antibody, zearalenone monoclonal antibody, T-2toxin monoclonal antibody and fumonisin B1And the SERS nano-probe is formed by coupling the monoclonal antibody and the vomitoxin monoclonal antibody.
2. The kit of claim 1, wherein the test strip is provided with a quality control line, and the quality control line is sprayed with a goat-anti-mouse secondary antibody.
3. The kit of claim 1 or 2, wherein 2 different antigens are coated on each of the 3 detection lines distributed on the detection strip in a mixed manner by combining:
aflatoxin M1An antigen and ochratoxin antigen;
zearalenone antigen and T-2toxin antigen;
fumonisins B1Antigens and vomitoxin antigens;
preferably, aflatoxin and ochratoxin antigens are coated at the detection line far away from the sample pad, zearalenone and T-2toxin antigens are coated at the middle detection line position, and fumonisin and vomitoxin are coated at the detection line position close to the sample pad.
4. The kit of claim 3, wherein, in the detection reagent,
with aflatoxin M1Raman markers of SERS nano probes coupled with the monoclonal antibody and the ochratoxin monoclonal antibody are different;
the Raman marker is different from the Raman marker of the SERS nano probe coupled with the zearalenone monoclonal antibody and the T-2toxin monoclonal antibody;
with fumonisins B1The Raman markers of the SERS nano-probe coupled with the monoclonal antibody and the vomitoxin monoclonal antibody are different.
5. The kit of claim 3, wherein the detection reagent binds to aflatoxin M1Raman markers of SERS nanoprobes coupled with the monoclonal antibody and the ochratoxin monoclonal antibody are DTNB and MBA respectively;
raman markers of the SERS nanoprobes coupled with the zearalenone monoclonal antibody and the T-2toxin monoclonal antibody are DTNB and MBA respectively;
with fumonisins B1Raman markers of the SERS nanoprobes coupled with the monoclonal antibody and the vomitoxin monoclonal antibody are DTNB and MBA respectively.
6. The kit of any one of claims 1 to 5, wherein the Au @ Ag core-shell nanoparticle complex has a particle size of 40 to 80nm, preferably a particle size of 52 nm.
7. The kit of any one of claims 1 to 6, wherein the OD of SERS nanoprobes of six mycotoxins in the detection reagent520Are all made of1.0, mixing the components in a volume ratio of 1:2:1:3:1:2 respectively, wherein the total volume is 200 mu L, and placing the mixture in a hole of an enzyme-labeled plate for freeze-drying for later use.
8. The kit of any one of claims 1-7, wherein the Au @ Ag core-shell nanoparticle complex is prepared by: 60 μ L of 200mM ascorbic acid and 15 μ L of 200mM AgNO were mixed at room temperature3Adding the mixture into a 10mL container of Au NPs with the particle size of about 30nm, and continuing to react for 30min under the stirring condition; addition of 60. mu.L of 200mM ascorbic acid and 15. mu.L of 200mM AgNO was continued3Reacting for 30 min; au @ Ag core-shell nanoparticle composites with the particle sizes of 48-56nm are prepared sequentially through 4 cycles, and the obtained particle composites are respectively centrifuged for 20min and re-dissolved in 10mL of ultrapure water.
9. The kit of claim 8, wherein the SERS nanoprobe is prepared by the steps of: mixing 10mLAu @ Ag core-shell nanoparticle complex with 500 μ L of 0.2M pH 8.5 boric acid buffer, adding 300 μ L of 1mM DTNB or 1mM MBA; the solutions are respectively slightly shaken at room temperature for reaction for 30min, centrifuged, redundant Raman molecules in supernatant are removed, and 10mL of 2.0mM boric acid buffer solution with pH of 8.5 is used for redissolving; respectively adding six mycotoxin antibodies into DTNB-Au @ Ag core-shell nanoparticle compound or MBA-Au @ Ag core-shell nanoparticle compound solution, and reacting for 1h under slow stirring; respectively adding 200 μ L of 1% PVP dissolved in 2.0mM boric acid buffer solution with pH of 8.5 for treatment, and masking bare sites on the surfaces of the nanoparticles to terminate the reaction; centrifuging the suspension at 3,400rpm for 10min, removing supernatant, re-dissolving, centrifuging, repeating twice, re-suspending the precipitate in 0.01M phosphate buffer containing 1% BSA at pH 7.4, and adjusting OD of each SERS nanoprobe520The value is 1.0, the mixture is mixed according to the volume ratio of 1:2:1:3:1:2 respectively, the total volume is 200 mu L, and the mixture is placed in a hole of an enzyme-labeled plate and is frozen and dried for standby.
10. SERS (surface enhanced Raman scattering) lateral flow immunosensing analysis method for detecting mycotoxin exposure marker in blood plasma or urine, wherein the mycotoxin is aflatoxin M1Ochratoxin, zearalenone, T-2toxin and fumonisin B1And vomitoxin; the method is characterized by comprising the following steps:
(1) pretreatment of a sample to be detected:
a) plasma sample pretreatment: taking 0.3mL of a plasma sample, adding 0.7mL of acetonitrile, fully whirling and mixing uniformly, centrifuging at 8000rpm for 10min, taking 0.5mL of supernatant, fully mixing with 2mL of PBS (phosphate buffer solution) containing 0.5% Triton-100 and 0.5% BSA (bovine serum albumin), and taking 200 mu L of the supernatant for SERS lateral flow immunosensing analysis; or
b) Pretreatment of a urine sample: centrifuging the urine sample at 6,000rpm for 5 minutes, uniformly mixing 0.5mL of supernatant with 0.5mL of PBS (phosphate buffer solution) containing 1% Triton and 1% BSA, and taking 200 mu L of supernatant for SERS lateral flow immunosensing analysis;
(2) carrying out a test sample detection using the kit of any one of claims 1 to 9: adding 200 mu L of sample solution into a detection reagent, uniformly mixing, incubating at room temperature for 3min, immersing the immune test strip into the incubated reagent, reacting for 10min, taking out the test strip, drying, and collecting SERS signals at the detection line by using a Raman spectrometer; the spectrometer parameters in the experiment were set as follows: the excitation light source adopts a He-Ne laser, the excitation wavelength is 785nm, the laser intensity is 20%, and the signal acquisition time is 5 s; separately record the DTNB labeled nanoprobes for 1332cm-1And 1589cm of MBA-labeled nano probe-1Signal strength of the band.
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